RESUMEN
BACKGROUND & AIMS: Studies are needed to determine the mechanism by which Barrett's esophagus (BE) progresses to esophageal adenocarcinoma (EAC). Notch signaling maintains stem cells in the gastrointestinal tract and is dysregulated during carcinogenesis. We explored the relationship between Notch signaling and goblet cell maturation, a feature of BE, during EAC pathogenesis. METHODS: We measured goblet cell density and levels of Notch messenger RNAs in BE tissues from 164 patients, with and without dysplasia or EAC, enrolled in a multicenter study. We analyzed the effects of conditional expression of an activated form of NOTCH2 (pL2.Lgr5.N2IC), conditional deletion of NOTCH2 (pL2.Lgr5.N2fl/fl), or loss of nuclear factor κB (NF-κB) (pL2.Lgr5.p65fl/fl), in Lgr5+ (progenitor) cells in L2-IL1B mice (which overexpress interleukin 1 beta in esophagus and squamous forestomach and are used as a model of BE). We collected esophageal and stomach tissues and performed histology, immunohistochemistry, flow cytometry, transcriptome, and real-time polymerase chain reaction analyses. Cardia and forestomach tissues from mice were cultured as organoids and incubated with inhibitors of Notch or NF-kB. RESULTS: Progression of BE to EAC was associated with a significant reduction in goblet cell density comparing nondysplastic regions of tissues from patients; there was an inverse correlation between goblet cell density and levels of NOTCH3 and JAG2 messenger RNA. In mice, expression of the activated intracellular form of NOTCH2 in Lgr5+ cells reduced goblet-like cell maturation, increased crypt fission, and accelerated the development of tumors in the squamocolumnar junction. Mice with deletion of NOTCH2 from Lgr5+ cells had increased maturation of goblet-like cells, reduced crypt fission, and developed fewer tumors. Esophageal tissues from in pL2.Lgr5.N2IC mice had increased levels of RelA (which encodes the p65 unit of NF-κB) compared to tissues from L2-IL1B mice, and we found evidence of increased NF-κB activity in Lgr5+ cells. Esophageal tissues from pL2.Lgr5.p65fl/fl mice had lower inflammation and metaplasia scores than pL2.Lgr5.N2IC mice. In organoids derived from pL2-IL1B mice, the NF-κB inhibitor JSH-23 reduced cell survival and proliferation. CONCLUSIONS: Notch signaling contributes to activation of NF-κB and regulates differentiation of gastric cardia progenitor cells in a mouse model of BE. In human esophageal tissues, progression of BE to EAC was associated with reduced goblet cell density and increased levels of Notch expression. Strategies to block this pathway might be developed to prevent EAC in patients with BE.
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Adenocarcinoma/patología , Esófago de Barrett/patología , Carcinogénesis/patología , Neoplasias Esofágicas/patología , Células Caliciformes/patología , Receptores Notch/metabolismo , Adenocarcinoma/diagnóstico , Adenocarcinoma/genética , Anciano , Animales , Esófago de Barrett/diagnóstico , Esófago de Barrett/genética , Biopsia , Carcinogénesis/genética , Diferenciación Celular/genética , Estudios Transversales , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Mucosa Esofágica/citología , Mucosa Esofágica/diagnóstico por imagen , Mucosa Esofágica/patología , Neoplasias Esofágicas/diagnóstico , Neoplasias Esofágicas/genética , Esofagoscopía , Femenino , Mucosa Gástrica/citología , Mucosa Gástrica/patología , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , FN-kappa B/metabolismo , Estudios Prospectivos , ARN Mensajero/análisis , ARN Mensajero/metabolismo , Receptores Notch/genética , Transducción de SeñalRESUMEN
BACKGROUND & AIMS: Barrett's esophagus (BE) is a precursor to esophageal adenocarcinoma (EAC). Progression from BE to cancer is associated with obesity, possibly due to increased abdominal pressure and gastroesophageal reflux disease, although this pathogenic mechanism has not been proven. We investigated whether environmental or dietary factors associated with obesity contribute to the progression of BE to EAC in mice. METHODS: Tg(ED-L2-IL1RN/IL1B)#Tcw mice (a model of BE, called L2-IL1B mice) were fed a chow (control) or high-fat diet (HFD) or were crossbred with mice that express human interleukin (IL) 8 (L2-IL1B/IL8 mice). Esophageal tissues were collected and analyzed for gene expression profiles and by quantitative polymerase chain reaction, immunohistochemistry, and flow cytometry. Organoids were established from BE tissue of mice and cultured with serum from lean or obese individuals or with neutrophils from L2-IL1B mice. Feces from mice were analyzed by 16s ribosomal RNA sequencing and compared to 16s sequencing data from patients with dysplasia or BE. L2-IL1B were mice raised in germ-free conditions. RESULTS: L2-IL1B mice fed an HFD developed esophageal dysplasia and tumors more rapidly than mice fed the control diet; the speed of tumor development was independent of body weight. The acceleration of dysplasia by the HFD in the L2-IL1B mice was associated with a shift in the gut microbiota and an increased ratio of neutrophils to natural killer cells in esophageal tissues compared with mice fed a control diet. We observed similar differences in the microbiomes from patients with BE that progressed to EAC vs patients with BE that did not develop into cancer. Tissues from dysplasias of L2-IL1B mice fed the HFD contained increased levels of cytokines that are produced in response to CXCL1 (the functional mouse homolog of IL8, also called KC). Serum from obese patients caused organoids from L2-IL1B/IL8 mice to produce IL8. BE tissues from L2-IL1B mice fed the HFD and from L2-IL1B/IL8 mice contained increased numbers of myeloid cells and cells expressing Cxcr2 and Lgr5 messenger RNAs (epithelial progenitors) compared with mice fed control diets. BE tissues from L2-IL1B mice raised in germ-free housing had fewer progenitor cells and developed less dysplasia than in L2-IL1 mice raised under standard conditions; exposure of fecal microbiota from L2-IL1B mice fed the HFD to L2-IL1B mice fed the control diet accelerated tumor development. CONCLUSIONS: In a mouse model of BE, we found that an HFD promoted dysplasia by altering the esophageal microenvironment and gut microbiome, thereby inducing inflammation and stem cell expansion, independent of obesity.
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Adenocarcinoma/patología , Esófago de Barrett/patología , Neoplasias Esofágicas/patología , Microbioma Gastrointestinal/fisiología , Interleucina-8/metabolismo , Obesidad/patología , Adenocarcinoma/inmunología , Adulto , Anciano , Animales , Esófago de Barrett/inmunología , Carcinogénesis/inmunología , Carcinogénesis/patología , Dieta Alta en Grasa/efectos adversos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Neoplasias Esofágicas/inmunología , Esófago/inmunología , Esófago/patología , Heces/microbiología , Femenino , Voluntarios Sanos , Humanos , Interleucina-8/genética , Interleucina-8/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Obesidad/sangre , Obesidad/inmunología , Organoides , Suero/inmunología , Suero/metabolismo , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
Risk stratification in patients with Barrett's esophagus (BE) to prevent the development of esophageal adenocarcinoma (EAC) is an unsolved task. The incidence of EAC and BE is increasing and patients are still at unknown risk. BarrettNET is an ongoing multicenter prospective cohort study initiated to identify and validate molecular and clinical biomarkers that allow a more personalized surveillance strategy for patients with BE. For BarrettNET participants are recruited in 20 study centers throughout Germany, to be followed for progression to dysplasia (low-grade dysplasia or high-grade dysplasia) or EAC for >10 years. The study instruments comprise self-administered epidemiological information (containing data on demographics, lifestyle factors, and health), as well as biological specimens, i.e., blood-based samples, esophageal tissue biopsies, and feces and saliva samples. In follow-up visits according to the individual surveillance plan of the participants, sample collection is repeated. The standardized collection and processing of the specimen guarantee the highest sample quality. Via a mobile accessible database, the documentation of inclusion, epidemiological data, and pathological disease status are recorded subsequently. Currently the BarrettNET registry includes 560 participants (23.1% women and 76.9% men, aged 22-92 years) with a median follow-up of 951 days. Both the design and the size of BarrettNET offer the advantage of answering research questions regarding potential causes of disease progression from BE to EAC. Here all the integrated methods and materials of BarrettNET are presented and reviewed to introduce this valuable German registry.
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Adenocarcinoma/diagnóstico , Esófago de Barrett/complicaciones , Detección Precoz del Cáncer/métodos , Neoplasias Esofágicas/diagnóstico , Vigilancia de la Población/métodos , Medición de Riesgo/métodos , Adenocarcinoma/etiología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores/análisis , Reglas de Decisión Clínica , Progresión de la Enfermedad , Neoplasias Esofágicas/etiología , Femenino , Alemania , Humanos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Sistema de Registros , Factores de Riesgo , Adulto JovenRESUMEN
A unique feature of the germinal center B cell-derived Hodgkin and Reed/Sternberg cells of classical Hodgkin lymphoma is their lost B cell phenotype and the aberrant expression of factors of other hematopoietic cell types, including ID2 and NOTCH1. As cellular dedifferentiation and upregulation of ID2 and NOTCH1 are typical consequences of a hypoxic response, we wondered whether hypoxia may impose an HRS cell-like phenotype in B cells. Culturing normal B cells or cell lines of germinal center-type diffuse large B-cell lymphoma under hypoxic conditions caused partial downregulation of several B cell markers, ID2 upregulation, and increased NOTCH1 activity. The hypoxic cells acquired further features of Hodgkin and Reed/Sternberg cells, including increased JUN expression, and enhanced NFκB activity. The Hodgkin and Reed/Sternberg cell-expressed epigenetic regulators KDM4C and PCGF2, as well as the phosphatase DUSP1 were partially induced in hypoxic B cells. Inhibition of DUSP1 was toxic for classical Hodgkin lymphoma cell lines. Thus, hypoxia induces key Hodgkin and Reed/Sternberg cell characteristics in mature B cells. We speculate that hypoxic conditions in the germinal center may impose phenotypic changes in germinal center B cells, promoting their survival and initiating their differentiation towards a Hodgkin and Reed/Sternberg cell-like phenotype. These may then be stabilized by transforming events in the Hodgkin and Reed/Sternberg precursor cells.
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Enfermedad de Hodgkin/etiología , Enfermedad de Hodgkin/metabolismo , Hipoxia/metabolismo , Linfocitos B/metabolismo , Linfocitos B/patología , Biomarcadores , Línea Celular Tumoral , Supervivencia Celular/genética , Epigénesis Genética , Expresión Génica , Centro Germinal/metabolismo , Centro Germinal/patología , Enfermedad de Hodgkin/patología , Humanos , Hipoxia/genética , Factor 1 Inducible por Hipoxia/genética , Factor 1 Inducible por Hipoxia/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfoma de Células B Grandes Difuso/etiología , Linfoma de Células B Grandes Difuso/metabolismo , Linfoma de Células B Grandes Difuso/patología , Estadificación de Neoplasias , FenotipoRESUMEN
Mesenchymal stromal cells (MSC) have been suggested to provide a suitable cellular environment for in vitro expansion of haematopoietic stem and progenitor cells (HPC) from umbilical cord blood. In this study, we have simultaneously analysed the cell division history and immunophenotypic differentiation of HPC by using cell division tracking with carboxyfluorescein diacetate N-succinimidyl ester (CFSE). Co-culture with MSC greatly enhanced proliferation of human HPC, especially of the more primitive CD34(+)CD38(-) fraction. Without co-culture CD34 and CD133 expressions decreased after several cell divisions, whereas CD38 expression was up-regulated after some cell divisions and then diminished in fast proliferating cells. Co-culture with MSC maintained a primitive immunophenotype (CD34(+), CD133(+) and CD38(-)) for more population doublings, whereas up-regulation of differentiation markers (CD13, CD45 and CD56) in HPC was delayed to higher numbers of cell divisions. Especially MSC of early cell passages maintained CD34 expression in HPC over more cell divisions, whereas MSC of higher passages further enhanced their proliferation rate. Inhibition of mitogen-activated protein kinase 1 (MAPK1) impaired proliferation and differentiation of HPC, but not maintenance of long-term culture initiating cells. siRNA knockdown of N-cadherin and VCAM1 in feeder layer cells increased the fraction of slow dividing HPC, whereas knockdown of integrin beta 1 (ITGB1) and CD44 impaired their differentiation. In conclusion, MSC support proliferation as well as self-renewal of HPC with primitive immunophenotype. The use of early passages of MSC and genetic manipulation of proteins involved in HPC-MSC interaction might further enhance cord blood expansion on MSC.
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Proliferación Celular , Células Madre Hematopoyéticas/fisiología , Células Madre Mesenquimatosas/fisiología , Células del Estroma/fisiología , ADP-Ribosil Ciclasa 1/metabolismo , Antígenos CD34/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Técnicas de Cultivo de Célula , Diferenciación Celular/fisiología , Células Cultivadas , Senescencia Celular/fisiología , Técnicas de Cocultivo , Sangre Fetal/citología , Células Madre Hematopoyéticas/citología , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/citología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Células del Estroma/citologíaRESUMEN
INTRODUCTION: Several systematic reviews and meta-analyses of primary studies have been published on the relationship between annual case load of carotid endarterectomy (CEA) and carotid artery stenting (CAS) performed at hospital level or by individual surgeons, and perioperative outcomes. Many studies on volume-outcome relationship have already been published and high-quality systematic reviews are crucial for further guideline development. EVIDENCE ACQUISITION: Systematic reviews and meta-analyses on the relationship between hospital or surgeon CEA/CAS volume and periprocedural outcomes were identified through a systematic literature search of Medline, Web of Science, and the Cochrane Database of Systematic Reviews. Methodological quality of the systematic reviews was appraised using the AMSTAR2 tool independently by two authors. Systematic reviews were aggregated in their volume-outcome findings. Quantitative data from primary studies included in the systematic reviews were synthesized. Additionally, volume definitions and the time point of outcome assessment used in primary studies were analyzed. EVIDENCE SYNTHESIS: In total, five systematic reviews published between 2000 and 2018 were identified, each comprising 11-25 primary studies. Methodological quality appraisal of these reviews revealed high quality for only the most recent review, low quality for three reviews, and critically low quality in one review. Aggregation of the systematic reviews revealed a significant inverse relationship between hospital/operator volume and the periprocedural risk of death or stroke following CEA. For CAS, high operator volume was associated with lower outcome rates. Regarding hospital volume, an inverse but non-significant relationship between CAS hospital volume and outcome rate was found. In our synthesis of primary studies from these systematic reviews an inverse CEA hospital and operator volume relationship was present for stroke or death and for CAS for hospital volume, respectively. A high heterogeneity regarding the definitions of volume categories, and of time points assessing outcomes was apparent. CONCLUSIONS: For CEA, high quality aggregated evidence revealed an inverse relationship between hospital/surgeon CEA volume and periprocedural rate of stroke or death. The same was true for operator linked CAS volume. Regarding hospital linked CAS volume, no unequivocal evidence was found. Additionally, heterogeneity was found regarding volume definition, and time of outcome assessment. Thus, future studies should aim to harmonize volume definitions and outcome time points.
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Enfermedades de las Arterias Carótidas/cirugía , Endarterectomía Carotidea/tendencias , Procedimientos Endovasculares/tendencias , Hospitales de Alto Volumen/tendencias , Hospitales de Bajo Volumen/tendencias , Pautas de la Práctica en Medicina/tendencias , Accidente Cerebrovascular/prevención & control , Cirujanos/tendencias , Enfermedades de las Arterias Carótidas/complicaciones , Enfermedades de las Arterias Carótidas/diagnóstico por imagen , Enfermedades de las Arterias Carótidas/mortalidad , Competencia Clínica , Endarterectomía Carotidea/efectos adversos , Endarterectomía Carotidea/mortalidad , Procedimientos Endovasculares/efectos adversos , Procedimientos Endovasculares/instrumentación , Procedimientos Endovasculares/mortalidad , Medicina Basada en la Evidencia , Humanos , Indicadores de Calidad de la Atención de Salud/tendencias , Medición de Riesgo , Factores de Riesgo , Stents/tendencias , Accidente Cerebrovascular/diagnóstico , Accidente Cerebrovascular/etiología , Accidente Cerebrovascular/mortalidad , Resultado del TratamientoRESUMEN
Mesenchymal stromal cells (MSC) provide a supportive cellular microenvironment and are able to maintain the self-renewal capacity of hematopoietic progenitor cells (HPC). Isolation procedures for MSC vary extensively, and this may influence their biologic properties. In this study, we have compared human MSC isolated from bone marrow (BM) using two culture conditions, from cord blood (CB), and from adipose tissue (AT). The ability to maintain long-term culture-initiating cell frequency and a primitive CD34(+)CD38(-) immunophenotype was significantly higher for MSC derived from BM and CB compared with those from AT. These results were in line with a significantly higher adhesion of HPC to MSC from BM and CB versus MSC from AT. We have compared the cytokine production of MSC by cytokine antibody arrays, enzyme-linked immunosorbent assay, and a cytometric bead array. There were reproducible differences in the chemokine secretion profiles of various MSC preparations, but there was no clear concordance with differences in their potential to maintain primitive function of HPC. Global gene expression profiles of MSC preparations were analyzed and showed that adhesion proteins including cadherin-11, N-cadherin, vascular cell adhesion molecule 1, neural cell adhesion molecule 1, and integrins were highly expressed in MSC preparations derived from BM and CB. Thus, MSC from BM and CB are superior to MSC from AT for maintenance of primitive HPC. The latter property is associated with specific molecular profiles indicating the significance of cell-cell junctions but not with secretory profiles. Disclosure of potential conflicts of interest is found at the end of this article.
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Células Madre Hematopoyéticas/citología , Mesodermo/citología , Células del Estroma/metabolismo , ADP-Ribosil Ciclasa 1/análisis , Tejido Adiposo/citología , Antígenos CD34/análisis , Adhesión Celular , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/genética , Comunicación Celular , Técnicas de Cultivo de Célula/métodos , Separación Celular , Células Cultivadas/metabolismo , Técnicas de Cocultivo , Citocinas/biosíntesis , Citocinas/metabolismo , Sangre Fetal/citología , Perfilación de la Expresión Génica , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/farmacología , Uniones Intercelulares/fisiología , Especificidad de ÓrganosRESUMEN
BACKGROUND: Direct cell-cell contact between hematopoietic progenitor cells (HPC) and their cellular microenvironment is essential for maintenance of 'stemness'. We have previously demonstrated that a feeder layer of human mesenchymal stromal cells (MSC) could provide a surrogate model as a niche for human HPC. Maintenance of long-term culture-initiating cells was significantly lower on fibroblasts. METHODS: Adhesion of HPC to MSC was further analyzed using our recently described adhesion assay based on gravitational force upon inversion and in combination with specific antibodies against CD44. RESULTS: Adhesion of KG1a and CD34+ cells was significantly reduced by administration of a monoclonal CD44 antibody and for KG1a to a greater extent than for CD34+ cells. Interaction of HPC and MSC was further analyzed by laser scanning confocal microscopy. CD44 was located on the uropod of CD34+ cells at the site of contact with MSC and both cell types were interwoven by a network of fibronectin. CONCLUSION: Various adhesion proteins, including CD44, are involved in the contact of human HPC and human MSC and further analysis of the relative significance and interaction of these proteins will be crucial for the understanding of the mechanism of this specific cell-cell interaction.
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Células Madre Hematopoyéticas/citología , Receptores de Hialuranos/metabolismo , Mesodermo/citología , Células del Estroma/citología , Anticuerpos Bloqueadores/farmacología , Antígenos CD34/metabolismo , Adhesión Celular/efectos de los fármacos , Comunicación Celular/efectos de los fármacos , Recuento de Células , Fibronectinas/metabolismo , Gravitación , Células Madre Hematopoyéticas/efectos de los fármacos , Humanos , Mesodermo/efectos de los fármacos , Microscopía Confocal , Células del Estroma/efectos de los fármacosRESUMEN
OBJECTIVE: The significant role of direct contact between hematopoietic progenitor cells (HPC) and the cellular microenvironment for maintaining "stemness" has been demonstrated. Human mesenchymal stem cell (MSC) feeder layers represent a surrogate model for this interaction. Specific adhesion molecules are responsible for this cell-cell contact. METHODS: To define cell-cell contact between HPC and MSC, we have studied adhesive interaction of various fractions of HPC by using a novel assay based on gravitational force upon inversion. Adherent and nonadherent cells were separated and further analyzed with regard to gene expression and long-term hematopoietic culture initiating cell (LTC-IC) frequency. RESULTS: HPC subsets with higher self-renewing capacity demonstrated significantly higher adherence to human MSC (CD34(+) vs CD34(-), CD34(+)/CD38(-) vs CD34(+)/CD38(+), slow dividing fraction vs fast dividing fraction). LTC-IC frequency was significantly higher in the adherent fraction than in the nonadherent fraction. Furthermore, genes coding for adhesion proteins and extracellular matrix were higher expressed in the adherent subsets of CD34(+) cells (fibronectin 1, cadherin 11, vascular cell adhesion molecule-1, connexin 43, integrin beta-like 1, and TGFBI). CONCLUSION: In this study we have demonstrated that primitive subsets of HPC have higher affinity to human MSC. The essential role of specific junction proteins for stabilization of cell-cell contact is indicated by their significant higher expression.
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Comunicación Celular/inmunología , Células Madre Hematopoyéticas/inmunología , Células Madre Mesenquimatosas/inmunología , Modelos Inmunológicos , Antígenos CD34/inmunología , Cadherinas/genética , Cadherinas/inmunología , Adhesión Celular/inmunología , Células Cultivadas , Conexina 43/genética , Conexina 43/inmunología , Proteínas de la Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/inmunología , Fibronectinas/genética , Fibronectinas/inmunología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Humanos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta/inmunología , Molécula 1 de Adhesión Celular Vascular/genética , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
OBJECTIVE: Mesenchymal stem cells (MSC) raise high hopes in clinical applications. However, the lack of common standards and a precise definition of MSC preparations remains a major obstacle in research and application of MSC. Whereas surface antigen markers have failed to precisely define this population, a combination of proteomic data and microarray data provides a new dimension for the definition of MSC preparations. METHODS: In our continuing effort to characterize MSC, we have analyzed the differential transcriptome and proteome expression profiles of MSC preparations isolated from human bone marrow under two different expansion media (BM-MSC-M1 and BM-MSC-M2). RESULTS: In proteomics, 136 protein spots were unambiguously identified by MALDI-TOF-MS and corresponding cDNA spots were selected on our "Human Transcriptome cDNA Microarray." Combination of datasets revealed a correlation in differential gene expression and protein expression of BM-MSC-M1 vs BM-MSC-M2. Genes involved in metabolism were more highly expressed in BM-MSC-M1, whereas genes involved in development, morphogenesis, extracellular matrix, and differentiation were more highly expressed in BM-MSC-M2. Interchanging culture conditions for 8 days revealed that differential expression was retained in several genes whereas it was altered in others. CONCLUSION: Our results have provided evidence that homogeneous BM-MSC preparations can reproducibly be isolated under standardized conditions, whereas culture conditions exert a prominent impact on transcriptome, proteome, and cellular organization of BM-MSC.
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Regulación de la Expresión Génica/fisiología , Células Madre Mesenquimatosas/fisiología , Proteoma/metabolismo , Transcripción Genética/fisiología , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/normas , Separación Celular/métodos , Separación Celular/normas , Células Cultivadas , Humanos , Células Madre Mesenquimatosas/citología , Proteoma/genética , Proteoma/normas , Reproducibilidad de los ResultadosRESUMEN
The cellular microenvironment in classical Hodgkin lymphoma (cHL) is dominated by a mixed infiltrate of inflammatory cells with typically only about 1% Hodgkin and Reed/Sternberg (HRS) tumor cells. T cells are usually the largest population of cells in the cHL microenvironment, encompassing T helper (Th) cells, regulatory T cells (Tregs), and cytotoxic T cells. Th cells and Tregs presumably provide essential survival signals for HRS cells. Tregs are also involved in rescuing HRS cells from antitumor immune responses. An understanding of the immune evasion strategies of HRS cells is not only relevant for a characterization of the pathophysiology of cHL but is also clinically relevant, given the current treatment approaches targeting checkpoint inhibitors. Here, we characterized the cHL-specific CD4+ T-cell infiltrate regarding its role in immune evasion. Global gene expression analysis of CD4+ Th cells and Tregs isolated from cHL lymph nodes and reactive tonsils revealed that Treg signatures were enriched in CD4+ Th cells of cHL. Hence, HRS cells may induce Treg differentiation in Th cells, a conclusion supported by in vitro studies with Th cells and cHL cell lines. We also found evidence for immune-suppressive purinergic signaling and a role of the inhibitory receptor-ligand pairs B- and T-cell lymphocyte attenuator-herpesvirus entry mediator and CD200R-CD200 in promoting immune evasion. Taken together, this study highlights the relevance of Treg induction and reveals new immune checkpoint-driven immune evasion strategies in cHL. Cancer Immunol Res; 5(12); 1122-32. ©2017 AACR.
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Enfermedad de Hodgkin/inmunología , Evasión Inmune , Línea Celular Tumoral , Perfilación de la Expresión Génica , Enfermedad de Hodgkin/genética , Enfermedad de Hodgkin/patología , Humanos , Evasión Inmune/genética , Evasión Inmune/inmunología , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/efectos de los fármacos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/efectos de los fármacos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Microambiente Tumoral/genética , Microambiente Tumoral/inmunologíaRESUMEN
PURPOSE: The therapy of unresectable advanced thyroid carcinomas shows unfavorable outcome. Constitutive nuclear factor-κB (NF-κB) activation in thyroid carcinomas frequently contributes to therapeutic resistance; the radioiodine therapy often fails due to the loss of differentiated functions in advanced thyroid carcinomas. Curcumin is known for its anticancer properties in a series of cancers, but only few studies have focused on thyroid cancer. Our aim was to evaluate curcumin's molecular mechanisms and to estimate if curcumin could be a new therapeutic option in advanced thyroid cancer. METHODS: Human thyroid cancer cell lines TPC-1 (papillary), FTC-133 (follicular), and BHT-101 (anaplastic) were treated with curcumin. Using real-time PCR analysis, we investigated microRNA (miRNA) and mRNA expression levels. Cell cycle, Annexin V/PI staining, and caspase-3 activity analysis were performed to detect apoptosis. NF-κB p65 activity and cell proliferation were analyzed using appropriate ELISA-based colorimetric assay kits. RESULTS: Treatment with 50 µM curcumin significantly increased the mRNA expression of the differentiation genes thyroglobulin (TG) and sodium iodide symporter (NIS) in all three cell lines and induced inhibition of cell proliferation, apoptosis, and decrease of NF-κB p65 activity. The miRNA expression analyses showed a significant deregulation of miRNA-200c, -21, -let7c, -26a, and -125b, known to regulate cell differentiation and tumor progression. Curcumin arrested cell growth at the G2/M phase. CONCLUSIONS: Curcumin increases the expression of redifferentiation markers and induces G2/M arrest, apoptosis, and downregulation of NF-κB activity in thyroid carcinoma cells. Thus, curcumin appears to be a promising agent to overcome resistance to the conventional cancer therapy.
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Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Puntos de Control del Ciclo Celular/efectos de los fármacos , Curcumina/farmacología , Neoplasias de la Tiroides/patología , Western Blotting , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Ensayo de Inmunoadsorción Enzimática , Perfilación de la Expresión Génica , Humanos , FN-kappa B/antagonistas & inhibidores , Reacción en Cadena en Tiempo Real de la Polimerasa , TranscriptomaRESUMEN
OBJECTIVE: Various preparative protocols have been proposed for the acquisition and cultivation of mesenchymal stem cells (MSC). Whereas surface antigen markers have failed to precisely define this population, microarray analysis might provide a better tool for characterization of MSC. METHODS: In this study, we have analyzed global gene expression profiles of human MSC isolated from adipose tissue (AT), from umbilical cord blood (CB), and from bone marrow (BM) under two growth conditions and have compared them to terminally differentiated human fibroblasts (HS68). Profiles were compared using our Human Genome Microarray representing 51.144 different cDNA clones. RESULTS: Cultured with the appropriate conditions, osteogenic and adipogenic differentiation could be confirmed in all MSC preparations but not in fibroblasts. No phenotypic differences were observed by flow cytometry using a panel of 22 surface antigen markers. Whereas MSC derived from different donors using the same culture procedure yielded a consistent and reproducible gene expression profile, many genes were differentially expressed in MSC from different ontogenetic sources or from different culture conditions. Twenty-five genes were overlapping and upregulated in all MSC preparations from AT, CB, and BM as compared to HS68 fibroblasts. These genes included fibronectin, ECM2, glypican-4, ID1, NF1B, HOXA5, and HOXB6. Many genes upregulated in MSC are involved in extracellular matrix, morphogenesis, and development, whereas several inhibitors of the Wnt pathway (DKK1, DKK3, SFRP1) were highly expressed in fibroblasts. CONCLUSION: Our results have provided a foundation for a more reproducible and reliable quality control using genotypic analysis for defining MSC.
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Linaje de la Célula , Perfilación de la Expresión Génica/normas , Células Madre Mesenquimatosas/citología , Tejido Adiposo/citología , Células de la Médula Ósea/citología , Técnicas de Cultivo de Célula , Diferenciación Celular , Sangre Fetal/citología , Fibroblastos/citología , Perfilación de la Expresión Génica/métodos , Humanos , Inmunofenotipificación , Células Madre Mesenquimatosas/metabolismo , Reproducibilidad de los ResultadosRESUMEN
The cellular microenvironment in HL is dominated by a mixed infiltrate of inflammatory cells with typically only 1 or a few percent of HRS tumor cells. HRS cells orchestrate this infiltrate by the secretion of a multitude of chemokines. T cells are usually the largest population of cells in the HL tissue, encompassing Th cells, T(regs), and CTLs. Th cells and T(regs) presumably provide essential survival signals for the HRS cells, and the T(regs) also play an important role in rescuing HRS cells from an attack by CTLs and NK cells. The interference with this complex interplay of HRS cells with other immune cells in the microenvironment may provide novel strategies for targeted immunotherapies.
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Enfermedad de Hodgkin/inmunología , Subgrupos de Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Animales , Comunicación Celular/efectos de los fármacos , Comunicación Celular/inmunología , Enfermedad de Hodgkin/metabolismo , Enfermedad de Hodgkin/patología , Enfermedad de Hodgkin/terapia , Humanos , Linfocitos Infiltrantes de Tumor/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , Escape del Tumor/inmunología , Microambiente Tumoral/efectos de los fármacosRESUMEN
Central to the formation of tissue at implant surfaces are the interactions between multiple cell types including fibroblasts, endothelial cells and, in the case of bone, cells of the osteoblastic lineage. To date the importance of population dynamics and interactions have been largely neglected in the in vitro evaluation of biomaterials. To fill this gap we have developed a co-culture system using 3 cell types, primary human bone marrow stromal cells (HBMC), microvascular endothelial cells (HMVEC) and abdominal dermal fibroblasts (HDF). Proliferation of each cell type separately and differentiation of HBMC were determined by flow cytometry analysis. The medium used promoted HBMC differentiation toward osteoblasts without affecting the state of differentiation of HDF and HMVEC. Furthermore, HBMC are strongly affected by HDF and HMVEC, and vice versa. When used on a titanium coated substrate the triple cell culture system identified preferential HBMC proliferation relative to HDF if HMVEC was present. This developed culture system represents a new, optimised and potentially predictive approach to evaluate biomaterial biocompatibility early in development.
Asunto(s)
Sustitutos de Huesos/química , Células Endoteliales/citología , Fibroblastos/citología , Ensayo de Materiales/instrumentación , Células Madre Mesenquimatosas/citología , Osteoblastos/citología , Osteogénesis/fisiología , Anciano , Anciano de 80 o más Años , Sustitutos de Huesos/análisis , Diferenciación Celular , Células Cultivadas , Técnicas de Cocultivo/instrumentación , Células Endoteliales/fisiología , Diseño de Equipo , Análisis de Falla de Equipo , Femenino , Fibroblastos/fisiología , Humanos , Masculino , Células Madre Mesenquimatosas/fisiología , Osteoblastos/fisiología , Análisis de Matrices Tisulares/instrumentaciónRESUMEN
The cellular origin of chronic lymphocytic leukemia (CLL) is still debated, although this information is critical to understanding its pathogenesis. Transcriptome analyses of CLL and the main normal B cell subsets from human blood and spleen revealed that immunoglobulin variable region (IgV) gene unmutated CLL derives from unmutated mature CD5(+) B cells and mutated CLL derives from a distinct, previously unrecognized CD5(+)CD27(+) post-germinal center B cell subset. Stereotyped V gene rearrangements are enriched among CD5(+) B cells, providing independent evidence for a CD5(+) B cell derivation of CLL. Notably, these CD5(+) B cell populations include oligoclonal expansions already found in young healthy adults, putatively representing an early phase in CLL development before the CLL precursor lesion monoclonal B cell lymphocytosis. Finally, we identified deregulated proteins, including EBF1 and KLF transcription factors, that were not detected in previous comparisons of CLL and conventional B cells.
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Subgrupos de Linfocitos B/metabolismo , Reordenamiento Génico de Linfocito B/genética , Región Variable de Inmunoglobulina/genética , Leucemia Linfocítica Crónica de Células B/etiología , Leucemia Linfocítica Crónica de Células B/fisiopatología , Linfocitosis/genética , Adulto , Análisis de Varianza , Antígenos CD5/metabolismo , Análisis por Conglomerados , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Leucemia Linfocítica Crónica de Células B/genética , Microscopía Fluorescente , Mutación/genética , Oligonucleótidos/genética , Análisis de Componente Principal , Transactivadores/metabolismo , Miembro 7 de la Superfamilia de Receptores de Factores de Necrosis Tumoral/metabolismoRESUMEN
Specific cell-cell junctions between hematopoietic stem cells (HSC) and their niche have been shown to regulate stem cell function. N-cadherin was suggested to play a central role in this process, whereas other studies indicated that it did not play an essential role in the murine model. We have analyzed the role of N-cadherin for interaction between hematopoietic progenitor cells (HPC) and supportive mesenchymal stromal cells (MSC) in a human-human setting. Expression of N-cadherin and of cadherin-11 (osteoblast cadherin) was analyzed in HPC by quantitative RT-PCR, Western blot, and flow cytometry. N-cadherin and cadherin-11 were expressed in HPC at a moderate level, whereas they were not detectable in differentiated cells. Confocal laser scanning microscopy revealed that N-cadherin and beta-catenin are colocalized at the junction of HPC and MSC. siRNA knockdown of N-cadherin or cadherin-11 as well as treatment with the blocking function antibody decreased adhesive interaction of HPC to MSC. Furthermore, knockdown of N-cadherin or blocking function antibody impaired maintenance of long-term culture-initiating cells (LTC-IC) on coculture of HPC and MSC. These results indicate that N-cadherin is involved in the bidirectional interaction of human HPC with their cellular determinants in the niche.
Asunto(s)
Cadherinas/metabolismo , Células Madre Hematopoyéticas/metabolismo , Células Madre Mesenquimatosas/metabolismo , Western Blotting , Cadherinas/genética , Células Cultivadas , Citometría de Flujo , Humanos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Stromal cell-derived factor-1alpha (SDF-1alpha) has pleiotropic effects on hematopoietic progenitor cells (HPCs). We have monitored podia formation, migration, proliferation, and cell-cell adhesion of human HPC under the influence of SDF-1alpha, a peptide agonist of CXCR4 (CTCE-0214), a peptide antagonist (CTCE-9908), and a nonpeptide antagonist (AMD3100). Whereas SDF-1alpha induced migration of CD34(+) cells in a dose-dependent manner, CTCE-0214, CTCE-9908, and AMD3100 did not induce chemotaxis in this concentration range albeit the peptides CTCE-0214 and CTCE-9908 increased podia formation. Cell-cell adhesion of HPC to human mesenchymal stromal cells was impaired by the addition of SDF-1alpha, CTCE-0214, and AMD3100. Proliferation was not affected by SDF-1alpha or its analogs. Surface antigen detection of CXCR4 was reduced upon treatment with SDF-1alpha or AMD3100 and it was enhanced by CTCE-9908. Despite the fact that all these molecules target the same CXCR4 receptor, CXCR4 agonists and antagonists have selective effects on different functions of the natural molecule.
RESUMEN
Cell-cell contact between stem cells and cellular determinants of the microenvironment plays an essential role in controlling cell division. Using human hematopoietic progenitor cells (CD34+/CD38-) and a stroma cell line (AFT024) as a model, we have studied the initial behavioral and molecular sequel of this interaction. Time-lapse microscopy showed that CD34+/CD38- cells actively migrated toward and sought contact with stroma cells and 30% of them adhered firmly to AFT024 stroma through the uropod. CD44 and CD34 are colocalized at the site of contact. Gene expression profiles of CD34+/CD38- cells upon cultivation with or without stroma for 16, 20, 48, or 72 hours were analyzed using our human genome cDNA microarray. Chk1, egr1, and cxcl2 were among the first genes upregulated within 16 hours. Genes with the highest upregulation throughout the time course included tubulin genes, ezrin, c1qr1, fos, pcna, mcm6, ung, and dnmt1, genes that play an essential role in reorganization of the cytoskeleton system, stabilization of DNA, and methylation patterns. Our results demonstrate directed migration of CD34+/CD38- cells toward AFT024 and adhesion through the uropod and that upon interaction with supportive stroma, reorganization of the cytoskeleton system, regulation of cell division, and maintenance of genetic stability represent the most essential steps.